The ultimate goal of this project is to determine a mechanism for alcohol-induced deficient fracture repair. Binge alcohol consumption is a contributing factor in up to 40% of all orthopaedic trauma cases, and excessive alcohol consumption is a risk factor for osteopenia and fracture-related complications such as delayed fracture healing and fracture nonunion. Of the millions of fractures occurring annually, 5-10% result in fracture non- union, which requires expensive and prolonged treatment and multiple surgical interventions. Alcohol-related bone loss is likely caused by disturbances created in the bone remodeling cycle, although the specific molecular pathways responsible for this targeted effect are not known. Canonical Wnt signaling is known to promote the formation of osteoblasts and chondrocytes from mesenchymal stem cells, a process necessary to achieve complete fracture repair. Key proteins of the canonical Wnt signaling pathway, 2-catenin and Lrp5, demonstrated decreased mRNA levels in a study performed in our laboratory. Tight regulation of 2-catenin levels have recently been shown to be crucial for fracture healing. Due to these observations, we hypothesize that binge alcohol exposure impairs fracture healing by disruption of canonical Wnt signaling during mesenchymal stem cell differentiation at the injury site. To elucidate the effects of binge alcohol on canonical Wnt signaling during fracture healing, the specific aims of this application are (i) To determine the effect of binge alcohol exposure on fracture callus strength, integrity, and cellular composition, (ii) To measure the cellular expression of canonical Wnt proteins following binge alcohol exposure in the callus, and (iii) To determine, in culture, the effects of alcohol exposure on canonical Wnt proteins during mesenchymal stem cell differentiation and to find a mechanism for alcohol's effects on osteoblast and chondrocyte differentiation. Aims (i) and (ii) will be carried out using a murine model of binge alcohol exposure with a stabilized tibial fracture. Fractured tibias will be utilized for immunohistochemistry of osteoblasts and chondrocytes, biomechanical strength testing, and evaluation of canonical Wnt protein expression using Western blot. In addition, a transgenic mouse will be employed to visualized activated 2-catenin signaling in the fracture callus to observe the effects of alcohol on canonical Wnt transcriptional activity. Aim (ii) will also attempt to reverse alcohol-induced deficient bone repair by introducing an adenovirus that expresses 2-catenin directly into the fracture callus. Aim (iii) will employ cell culture techniques to push stem cells toward an osteoblast or adipocyte lineage with alcohol treatment, while testing the specific effects of alcohol of canonical Wnt mRNA expression during the differentiation process. Overall, this application will potentially reveal therapeutic targets that can be used to improve fracture healing and other musculoskeletal injuries in alcohol-abusing patients. PUBLIC HEALTH RELEVANCE: Fracture non-union and delayed union are complications arising in a higher frequency among the alcohol- abusing population, and are a significant healthcare burden. This application will potentially provide a link between fracture complications and alcohol abuse, and reveal therapeutic targets to aid in better care of musculoskeletal injuries.